Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response

While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.     

3D-QSAR studies of checkpoint kinase 1 inhibitors based on molecular docking and CoMFA

Three-dimensional quantitative structure–activity relationship (3D-QSAR) studies were performed on a series of substituted 1,4-dihydroindeno[1,2-c]pyrazoles inhibitors, using molecular docking and comparative molecular field analysis (CoMFA). The docking results from GOLD 3.0.1 provide a reliable conformational alignment scheme for the 3D-QSAR model. Based on the docking conformations and alignments, highly predictive CoMFA model was built with cross-validated q ² value of 0.534 and non-cross-validated partial least-squares analysis with the optimum components of six showed a conventional r ² value of 0.911. The predictive ability of this model was validated by the testing set with a conventional r ² value of 0.812. Based on the docking and CoMFA, we have identified some key features of the 1,4-dihydroindeno[1,2-c]pyrazoles derivatives that are responsible for checkpoint kinase 1 inhibitory activity. The analyses may be used to design more potent 1,4-dihydroindeno[1,2-c]pyrazoles derivatives and predict their activity prior to synthesis.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

Asymmetric Zinc‐Catalyzed Hydrosilylation of Ketones and the Effect of Carboxylate on the Enantioselectivity

Several chiral ligands containing (R,R)‐diaminocyclohexane moieties and pyrrole, furan, or benzene have been synthesized. These ligands were tested in enantioselective zinc‐catalyzed hydrosilylation reactions; excellent enantioselectivities were obtained when the ligands containing (R,R)‐diaminocyclohexane moieties and furan rings were used. For comparison, zinc chloride combined with different potassium carboxylate salts and ligands were also tested for catalytic hydrosilylation reactions. Chirality 25:275–280, 2013. © 2013 Wiley Periodicals, Inc.     

Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.     

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10⁵ CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.     

Design and synthesis of novel D-ring fused steroidal heterocycles

Using dehydroepiandrosterone as the starting material, we have synthesized a series of steroid analogs possessing a D-ring fused with heterocycles which are pyridine, imidazo [2,1-b]thiazoles or substituted thiazole imines. All the final structures are first reported and identified by NMR and MS spectroscopys, the yields of these products are moderate to good and the reaction conditions are mild. The cytotoxicity of the synthesized compounds against EC-109(human esophageal carcinoma), EC-9706(human esophageal carcinoma), MGC-803(human gastric carcinoma) were investigated.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.